FRAP Protocol

www.stke.org/cgi/content/full/sigtrans;2004/238/pl10 Page 1

INTRODUCTION

MATERIALS

Cell Culture Reagents and SuppliesCell LinesPurified Chaperone and Cochaperone ProteinsChemicalsFluorescent Protein Tags and Expression Vectors

EQUIPMENT

Cell CultureTemperature ControlMicroscopeQuantitative Image Analysis

RECIPES

INSTRUCTIONS

Biochemical Permeabilization and ExtractionIn Situ Nuclear Mobility Factor AssayFRAP ParametersQuantitative FRAP Analysis

NOTES AND REMARKS

REFERENCES AND NOTES

A Novel In Situ Assay for the Identification andCharacterization of Soluble Nuclear Mobility

FactorsCem Elbi,1 Dawn A. Walker,1 Marcia Lewis,2 Guillermo Romero,2 William P. Sullivan,3

David O. Toft,3 Gordon L. Hager,1* and Donald B. DeFranco2*

(Published 22 June 2004)

(Revised 6 July 2004)

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1Laboratory of Receptor Biology and Gene Expression, Building 41, Room B602, National Cancer Institute, Bethesda, MD 20892–5055, USA.2Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. 3Department of Biochemistry andMolecular Biology, Mayo Graduate School, Rochester, MN 55905, USA.

*Corresponding authors: E-mail, [email protected] (G.L.H.); [email protected] (D.B.D.)


Abstract

The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniqueshave revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged proteincan be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching(FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus.However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absenceof protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay thatcombines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a methodwe used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors.This assay can readily be adapted to identify and characterize other nuclear mobility factors.

Introduction

The visualization and precise quantitation of protein mobility in live cells through the use of photobleaching techniques has led toan increased understanding of the mechanisms responsible for intracellular protein trafficking (1, 2). Although protein mobilitywithin the nucleus can approach the limits predicted for the diffusion of free solute, low- or high-affinity interactions with specificsoluble or solid-state targets can reduce the kinetics of nuclear protein trafficking (3). However, even nuclear proteins that partici-pate in high-affinity interactions with large macromolecular complexes are highly mobile and possess diffusion coefficients that canapproach 0.5 µm2 s−1, as measured by photobleaching techniques in live cells (3).

In recent years, many green fluorescent protein (GFP) chimeras of nuclear proteins have been assessed for their localization within specificsubnuclear compartments and their intranuclear mobility. One family of nuclear proteins that has been the subject of particular scrutiny inthis regard is the nuclear receptor (NR) superfamily. NR proteins participate in various physiological responses to small ligands such as hor-mones, vitamins, and the metabolites of steroids, bile acids, and fatty acids (4, 5). NRs regulate the transcription of unique sets of targetgenes, usually in response to ligand binding (4, 5). NRs associate with their target genes through direct interactions with specific DNA se-quences or tethering to other DNA-bound factors, resulting in the recruitment of large co-regulator complexes (6, 7). These co-regulators in-clude coactivator and co-repressor complexes that modulate RNA polymerase II activity, in part through direct modification of histone pro-teins within core nucleosomes (8–10). In some cases, the mobility of NR co-regulator proteins has been examined in live cells. For example,nuclear mobility of the glucocorticoid receptor-interacting protein-1 (GRIP-1) coactivator is reduced when it interacts with the ligand-boundglucocorticoid receptor (GR) on target genes in live cell nuclei, implying that the assembly of functional complexes of NRs and coactivatorson active promoters provides one type of scaffold that may limit nuclear protein mobility (11).

Despite the increasing number of nuclear proteins assessed for mobility in live cells, our knowledge of the factors that regulate protein traf-ficking and mobility within the nucleus is limited. Both biochemical and live-cell imaging experiments reveal that the assembly and disas-sembly of large nuclear receptor-coactivator complexes on active genes is very rapid (12, 13), yet we know little about the mechanisms thatensure the efficient turnover of such supramolecular assemblies. In many paradigms of protein trafficking, model in situ systems have beeninstrumental in the identification of both specific targeting signals and soluble transport factors that function in interorganelle protein traffick-ing. Here, we describe an in situ assay for nuclear protein mobility in which the effects of specific subnuclear trafficking factors can be as-sessed. Importantly, cells analyzed with this assay maintain their transcriptional competence.

Digitonin has been used extensively as a permeabilization agent that maintains both transcriptional competence and the energy- and trans-port factor-dependent trafficking of proteins between the cytoplasm and nucleus (14, 15). However, most proteins added to digitonin-perme-abilized cells will not enter the nucleus unless specific nuclear transport factors are also included to permit their transit through the intact nu-clear pore complex. This feature of the digitonin-permeabilized cells limits their usefulness as an assay system to characterize the effects ofexogenously added factors on any nuclear function (for example, nuclear mobility). It was therefore necessary to develop additional proto-cols for permeabilization or extraction or both that would partially disrupt the nuclear pore complex to allow exogenously added proteins togain access to the nucleus yet maintain nuclear integrity. Previously, we identified a hypotonic buffer treatment of digitonin-permeabilizedcells that was efficient at extracting unliganded GR from nuclei, yet incapable of significantly depleting the nucleus of ligand-bound GR(16). We therefore reasoned that this combination of digitonin-permeabilization and hypotonic buffer extraction might allow for selective de-pletion of some nuclear proteins and not lead to the irreversible disruption of essential nuclear functions.

We applied a digitonin-permeabilization plus hypotonic buffer extraction protocol in mouse 3617.4 mammary adenocarcinoma cellline (17) to investigate the roles of putative subnuclear trafficking factors in influencing the localization and mobility of GFPchimeras of GR and PR-B. Mouse 3617.4 and 5953 cells contain an integrated copy of a GFP-GR and GFP-PR-B chimera under thecontrol of a tetracycline (tet)-regulated promoter (18,19). As will be shown below, in 3617.4 cells subjected to our permeabilizationand extraction protocol, nuclear GFP-GR chimeras are rendered immobile as assessed by fluorescence recovery after photobleach-ing (FRAP) analysis. Therefore, the permeabilization and extraction of 3617.4 and 5953 cells provided a useful system to identifyand characterize putative subnuclear trafficking factors that could be revealed by their ability to recover GFP-GR and GFP-PR-Bmobility. We have found that this procedure can easily be adapted to other cell types for the analysis of nuclear mobility of otherproteins.

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Materials

Cell Culture Reagents and Supplies

Charcoal-stripped fetal bovine serum (Hyclone, #SH30068.03)

Dulbecco’s modified eagle medium (DMEM; Gibco, #11960)

Fetal bovine serum (FBS; Atlanta Biologicals, #S11150)

Hanks’ balanced salt solution (HBSS; Gibco, #14025)

L-Glutamine, 100× (Invitrogen, #25030)

Nonessential amino acids, 100× (Invitrogen, #11140)

Penicillin-streptomycin, 100× (Invitrogen, #15140)

Phosphate-buffered saline (PBS; Quality Biologicals, #114-057-100)

Rabbit reticulocyte lysate (Promega, #L4960)

Sodium pyruvate, 100× (Invitrogen, #11360)

Tissue culture dishes, 35 mm and 100 mm (Falcon, #353001 and 353003)

Trypan blue dye, 0.4% (Invitrogen, #15250)

Trypsin in 1 mM ethylenediamine tetraacetic acid (EDTA) (Invitrogen, #25200)

Two-chambered Labtek II coverglass (Nalgene, #155360)

Cell LinesNote: The assay described in this Protocol can be performed with any cell line expressing GFP fusion protein stably.

C127 mouse fibroblast derivatives

Mouse mammary adenocarcinoma cell line (3617.4) stably expressing GFP-rat GR (18)

Mouse mammary adenocarcinoma cell line (5953) stably expressing enhanced GFP-human progesterone receptor (PR)-B

Purified Chaperone and Cochaperone Proteins Note: See (20) for details on purification unless otherwise noted.

Human Hsp70 (recombinant form prepared in Sf9 cells)

Human Hop (p60) (recombinant form prepared in bacteria)

Human CHIP [as described in (17), recombinant His-tagged CHIP expressed in bacteria was purified using a Stratagene Talon column]

Human FKBP51 (recombinant form prepared in bacteria)

Human Hsp90β (recombinant form prepared in Sf9 cells)

Human p23 (recombinant form prepared in bacteria)

Yeast Ydj-1 (recombinant form prepared in bacteria)

Chemicals

Adenosine triphosphate, disodium salt (ATP)

Bovine serum albumen (BSA)

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Creatine phosphate

Creatine phosphokinase

Dexamethasone (Sigma, #D-1756)

Digitonin

Note: Some vendors do not supply digitonin at 100% purity. The purity of digitonin is indicated in the chemical specificationsheet supplied by the vendor. Thus, always check purity of digitonin and vary the amount added if necessary.

Dimethyl sulfoxide (DMSO)

Dithiothreitol (DTT)

Ethanol, 200-proof

Ethylene glycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA)

G418 (Gibco, #11811)

Hepes, 1 M (pH 7.3) (Quality Biologicals, #118-089-060)

Magnesium acetate

Magnesium chloride, 1 M (Quality Biologicals, #351-033-060)

Potassium acetate (Quality Biologicals, #351-035-060)

Potassium chloride, 1 M (Quality Biologicals, #351-044-100)

R5020 (NEN Life Science, #NLP-004)

Sodium acetate

Sodium acetate, 3 M (pH 5.2) (Quality Biologicals, #351-035-060)

Tetracycline (Fisher, #BP912-100)

Triton X-100

Fluorescent Protein Tags and Expression Vectors

Green fluorescent protein (GFP) and Enhanced GFP (Clontech)

Tet-Off inducible system (Invitrogen): pTet-Splice, pTet-tTAK, and pTKneo

Equipment

Cell Culture

Hemacytometer

Tissue culture incubator at 37°C, 5% CO2 (Forma Scientific)

Temperature Control

ASI 400 Air Stream incubator (Nevtek)

Microscope

Confocal microscope LSM 510 (Zeiss) equipped with 40-mW argon laser

63× or 100× 1.3 numerical aperture oil immersion objective

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Quantitative Image Analysis

LSM 510 image analysis software (Zeiss)

Microsoft Excel or any other spread sheet software

Recipes

Recipe 1: Complete Culture MediumDMEM 430 ml

FBS 50 ml

Nonessential amino acids, 100× 5 ml

Sodium pyruvate, 100× 5 ml

L-Glutamine, 100× 5 ml

G418, 300 mg/ml stock 1.6 ml

Penicillin-streptomycin, 100× 5 ml

Dissolve 3.72 g of G418 in 11.4 ml of water and 1 ml of 1 M Hepes, pH 7.3 to make a 300-mg/ml stock solution. Filter sterilize andstore at 4°C.

Dissolve 500 mg of tetracycline in 50 ml of 200-proof ethanol and store in the dark at −20°C. Immediately before changing themedium, add 50 µl of this 10 mg/ml tetracycline to 500 ml of Complete Culture Medium for a final concentration of 10 µg/ml.

Complete culture medium containing tetracycline must be used immediately.

Note: Cells expressing GFP-GR or GFP-PR-B under the control of a tet-regulated promoter are maintained in this medium tosuppress GFP-GR or GFP-PR-B expression.

Recipe 2: Tetracycline-Free Culture MediumDMEM 430 ml

FBS 50 ml






Store this medium at 4°C for 20 days.

Recipe 3: Tetracycline-Free Culture Medium with Charcoal-Stripped FBSDMEM 430 ml

Charcoal-stripped FBS 50 ml






Store this medium at 4°C for 20 days.

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Recipe 4: 100 µM DexamethasoneDissolve 3.925 g of dexamethasone in 10 ml of ethanol to make a 1 M stock solution. Dilute this stock in ethanol to make a 100-µMstock solution, and store at −20°C. Chill dexamethasone on ice before use.

Note: Dexamethasone is used at a working concentration of 100 nM to stimulate GR.

Recipe 5: 30 µM R5020Dissolve 5 mg of R5020 in 1.53 ml of ethanol to make a 10-mM stock. Dilute this stock in ethanol to make a 30-µM stock solution,and store at −20°C. Chill R5020 on ice before use.

Note: R5020 is used at a working concentration of 30 nM to stimulate PR.

Recipe 6: Transport Buffer

Reagent Amount Final concentration

1 M Hepes-KOH, pH 7.8 10 ml 20 mM, pH 7.8

1 M Potassium acetate 55 ml 110 mM

1 M Sodium acetate 2.5 ml 5 mM

1 M Magnesium acetate 1 ml 2 mM

0.5 M EGTA, pH 8.0 1 ml 1 mM

Dissolve 238.3 g of Hepes in 1000 ml of deionized water to make a 1 M stock solution. Titrate to pH 7.8 with 1 M KOH (56.1 g ofKOH dissolved in 1000 ml of deionized water).

Dissolve 214.5 g of magnesium acetate in 1000 ml of deionized water to make a 1 M stock solution.

Dissolve 190.2 g of EGTA in 1000 ml of deionized water final volume to make a 0.5 M stock solution. Titrate to pH 8.0 with 1 M KOH.

Dissolve 98.2 g of potassium acetate in 1000 ml of deionized water to make a 1 M stock solution.

Dissolve 136.1 g of sodium acetate in 1000 ml of deionized water to make a 1 M stock solution.

Prepare Transport Buffer in 500 ml of water, filter sterilize, and store at 4°C. Chill on ice before use.

Recipe 7: Permeabilization BufferDissolve 40 mg of digitonin in 1 ml of DMSO to make a 40 mg/ml stock solution and store at room temperature shielded from light. Add 5 µlof this 40 mg/ml digitonin stock to 10 ml of Transport Buffer (Recipe 6) just before use, for a final concentration of 20 µg/ml.

Dissolve 154.3 mg of DTT in 1 ml of deionized water to make a 1 M stock solution. Store at −20°C. Add 10 µl of this 1M DTT stockto 10 ml of Transport Buffer (Recipe 6) just before use, for a final concentration of 1 mM. Chill on ice before use.

Recipe 8: Wash Buffer ADissolve 10 mg of BSA in 1 ml of deionized water to make a 10 mg/ml stock solution. Filter-sterilize and store at 4°C. Add 10 µl of this 10mg/ml BSA stock to 10 ml of Transport Buffer (Recipe 6) just before use, for a final concentration of 10 µg/ml. Chill on ice before use.

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Recipe 9: Hypotonic Extraction Buffer


1 M Hepes-KOH, pH 7.8 5 ml 10 mM

1 M Potassium chloride 5 ml 10 mM

1 M Magnesium chloride 0.75 ml 1.5 mM

Triton X-100 0.25 ml 0.05%

1 M DTT 10 µl 1 mM

Prepare Hypotonic Extraction Buffer in 500 ml of deionized water without adding DTT; filter-sterilize and store at 4°C.

Just before use, add 10 µl of 1M DTT stock to 10 ml of Hypotonic Extraction Buffer, for a final concentration of 1 mM. Chill on icebefore use.

Recipe 10: Recovery Buffer


1 M Hepes-KOH, pH 7.8 0.2 ml 20 mM, pH 7.8

1 M Potassium acetate 1.1 ml 110 mM

1 M Sodium acetate 50 µl 5 mM

1 M Magnesium acetate 20 µl 2 mM

0.5 M EGTA 20 µl 1 mM

BSA 200 mg 20 mg/ml

1 M DTT* 10 µl 1 mM

0.3 M ATP* 167 µl 5 mM

0.5 M Creatine phosphate* 100 µl 5 mM

2 U/µl Creatine phosphokinase* 100 µl 0.02 U/µl

Dissolve 1.8 g of ATP in 10 ml of PBS to make a 0.3 M stock solution. Store in 500 µl aliquots at −20°C.

Dissolve 1.6 g of creatine phosphate in 10 ml of deionized water to make a 0.5 M stock solution. Store in 500 µl aliquots at −20°C.

Dissolve 200 U of creatine phosphokinase in 100 µl of deionized water to make a 2 U/µl stock solution. Store in 10 µl aliquots at −20°C.

Prepare Recovery Buffer in 10 ml of deionized water without adding DTT or the components of ATP regeneration system marked with an *.Filter sterilize and store at 4°C. Add components of ATP regenerating system and DTT just before use. Chill on ice before use.

Note: When necessary, rabbit reticulocyte lysate or purified chaperones and cochaperones are added to Recovery Buffer just before use.

Instructions

Biochemical Permeabilization and Extraction

The following procedure has been designed for use with derivatives of C127 mouse fibroblasts and mouse mammary adenocarci-noma cell lines stably expressing GFP-GR (3617.4 cell line) or GFP-PR-B (5953 cell line). Addition of tetracycline to thegrowth medium is not necessary if a tetracycline-regulated system is not used. Moreover, replacement of medium with growthmedium containing charcoal-stripped FBS is not necessary if the experiments do not involve steroid receptors or other proteinsresponsive to hormones found in FBS.

The critical parameters that influence the extent of permeabilization and nuclear protein extraction are the concentration of digi-tonin in the permeabilization buffer, the concentration of Triton X-100 in the extraction buffer, the duration of the incubationswith digitonin- and Triton X-100-containing buffers, and the metabolic rate and density of the cells. These parameters will varydepending upon the cell type and must be optimized for successful permeabilization and extraction (see Notes and Remarks).Recommended initial concentration ranges are 5 to 50 µg/ml for digitonin and 0.01% to 0.5% for Triton X-100. Recommendedranges of incubation times are 2 to 10 min with digitonin-containing buffer and 0.5 to 5 min with Triton X-100-containing buffer.

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1. Grow cells in Complete Culture Medium (Recipe 1).

2. Plate 40,000 cells in each chamber of a Labtek II two-chambered coverglass along with 1 ml of Tetracycline-Free CultureMedium (Recipe 2). Incubate for 24 hours at 37°C, 5% CO2.

Note: If cells will be processed for biochemistry protocols (such as a Western blot), rather than for FRAP analysis as de-scribed here, plate 70,000 cells in 2 ml of medium per 35 mm plate. If a different size of tissue culture dish is used, scale thecell number and culture medium volumes proportionally.

3. Remove all media and wash the cells three times with 1 ml of HBSS per wash.

Note: When changing solutions or washing cells, completely remove old solution by tilting the Labtek II two-chamberedcoverglass and aspirating the pooled solution with a glass capillary pipette.

4. Add 1 ml of Tetracycline-Free Culture Medium with Charcoal-Stripped FBS (Recipe 3) to each Labtek II chamber. Placecells in incubator at 37°C, 5% CO2 for at least 16 hours (or overnight).

5. Add 100 µM Dexamethasone (Recipe 4) at 1:1000 for a final concentration of 100 nM, or 30 µM R5020 (Recipe 5) at1:1000 for a final concentration of 30 nM. Incubate for 1 hour.

Note: Dexamethasone and R5020 are used to stimulate translocation of GFP-GR and GFP-PR-B to the nucleus, respective-ly. This step is not necessary if the experiments do not involve investigation of steroid receptors.

6. Place Labtek II chambered coverglass on ice to keep cells at 4°C.

Note: Keep them on ice for the remaining steps in this procedure (steps 7 through 14).

7. Gently rinse the cells once with 2 ml of ice-cold PBS per Labtek II chamber.

8. Gently rinse the cells once with 2 ml of ice-cold Transport Buffer (Recipe 6) per chamber. Completely aspirate the bufferwith a glass capillary pipette.

9. Add 1 ml of ice-cold Permeabilization Buffer (Recipe 7) into each chamber and incubate the cells on ice for 5 min.

10. Remove Permeabilization Buffer and rinse cells once with 2 ml of ice-cold Transport Buffer (Recipe 6) per chamber.

11. Remove Transport Buffer and rinse cells once with 2 ml of ice-cold Wash Buffer A (Recipe 8) per chamber.

Note: These permeabilized cells can be maintained for up to 30 min on ice in 2 ml of Wash Buffer A without any obviousloss of nuclear GFP-GR protein. However, for in situ mobility assays, we recommend that users immediately proceed tostep 12, because the stability of other nuclear proteins with long-term storage on ice has not been tested.

12. For hypotonic extraction, remove Wash Buffer A and add 2 ml of ice-cold Hypotonic Extraction Buffer (Recipe 9) to eachchamber and incubate the cells on ice for 2 min.

13. Rinse the cells twice with 2 ml of ice-cold Transport Buffer (Recipe 6) per chamber.

14. Rinse the cells once with 2 ml of ice-cold Wash Buffer A (Recipe 8) per chamber.

Note: Extracted cells can be kept in Wash Buffer A on ice (up to 30 min if necessary), without any obvious loss of nuclear GFP-GRprotein, while awaiting further treatments. However, we recommend that users immediately proceed to the in situ nuclear mobilityfactor assay, because the stability of other nuclear proteins to long-term storage on ice has not been tested.

In Situ Nuclear Mobility Assay

Recovery conditions with either added reticulocyte lysate or purified molecular chaperones were also optimized for derivativesof C127 mouse fibroblasts and mouse mammary adenocarcinoma cell lines stably expressing GFP-GR (3617.4) or GFP-PR-B(5953). Recovery conditions were adapted from parameters established in experiments analyzing the recovery of hormone-bind-ing activity of steroid receptor heteromeric complexes (20, 21). Different buffer components, concentrations, and incubation con-ditions may be optimal for other nuclear mobility factors or cell types. Thus, these parameters will vary depending upon cell typeand must be optimized to develop a successful in situ mobility assay (see Notes and Remarks).

1. Remove Wash Buffer A completely (added in step 14 above) by tilting the Labtek II two-chambered coverglass and aspirat-ing the buffer with a glass capillary pipette.

2. Add 0.25 ml of Recovery Buffer (Recipe 10) to each Labtek II chamber. Place the cells in tissue culture incubator set at37°C, 5% CO2 for 10 min.

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Note: To test for ATP-dependent recovery of nuclear mobility in the absence of exogenously added factors, proceed immedi-ately to FRAP Parameters. To test for the recovery of nuclear mobility with reticulocyte lysate or with purified chaperoneand cochaperone proteins, follow step 3 or step 6, respectively.

3. Add 50 µl of rabbit reticulocyte lysate to 200 µl of Recovery Buffer (Recipe 10).

4. Gently add 250 µl of the lysate-buffer mix to each chamber.

5. Incubate the cells for 10 min at 37°C, 5% CO2.

Note: Reticulocyte lysate can be preincubated for 5 min at 37∞C with compounds that inhibit the activity of specific compo-nents (for instance, geldanamycin inhibits Hsp90 function) before incubating the mixture with permeabilized and extractedcells.

6. Add purified chaperone or cochaperone proteins to Recovery Buffer (Recipe 10).

Note: Purified chaperone or cochaperone proteins are used in the following amounts per 250 µl of Recovery Buffer (Recipe10) in the recovery assay: 20 µg of Hsp70, 2 µg of Ydj-1, 5 µg of Hop, 20 µg of Hsp90, 5 µg of p23, 10 µg of FKBP51, and10 µg of CHIP.

7. Gently add 250 µl of chaperone or cochaperone and buffer mix to each chamber.

8. Incubate the cells for 10 min at 37°C, 5% CO2.

Note: Cells are maintained at 37°C after the recovery period and during FRAP analysis. Cells are processed for FRAPanalysis immediately after the 10-min recovery period.

FRAP Parameters

In FRAP experiments, the GFP-GR (or GFP-PR-B) fluorescence signal in a small region of the nucleus is irreversibly photo-bleached by a short and intense laser pulse. Immediately after the bleach, the recovery of the GFP-GR (or GFP-PR-B) fluores-cence signal in the bleached area is monitored by fast sequential imaging. The signal in the bleached area is measured and thefluorescence recovery curve (normalized fluorescence intensity as a function of time) is generated by image analysis software.Images are acquired until the FRAP recovery is complete (until the quantitative FRAP recovery curve shows a plateau). Use theleast number of images and the longest time intervals that are acceptable to minimize photobleaching due to imaging. Photo-bleaching due to monitoring should result in less than 10% decrease in the fluorescence signal intensity as compared to pre-bleach value.

To compare individual data sets obtained under various experimental conditions or on different days, users must employ exactlythe same FRAP settings. Thus, magnification and zoom settings can be modified according to the size of the cell and the regionof interest, but the same settings must be used in different experiments to compare individual data sets without correcting for thesize of the bleached area.

Initially, test the efficiency and the depth of bleach on a fixed sample [samples can be fixed in 2% paraformaldehyde for 20 minat room temperature as in (22)]. In the fixed sample, the average fluorescence intensity in the bleached region should be less than30% of that in the unbleached region. If necessary, increase the number of short bleach pulses. The size of a nuclear bleach re-gion influences the quantitative FRAP analysis. Depending on the imaging area of interest, the size of a bleach region may vary.We have successfully used bleach regions in the size of a small circular spot or a 2- to 4-µm strip across the width of a cell.

The following FRAP procedure has been designed for use on the Zeiss LSM 510 confocal microscope. In-depth discussions ofvarious photobleaching techniques are available (23, 24).

1. Place the Labtek II two-chambered coverglass containing the permeabilized and extracted cells onto the stage of a confocalmicroscope.

Note: Maintain cell temperature at 37°C with an air stream incubator during image acquisition.

2. Use an oil immersion lens with 63× or 100× magnification.

3. Adjust laser output from a 40-mW argon laser and choose a small pinhole diameter of 1 Airy unit.

4. Find a cell of interest, set the zoom, and capture image with maximum image acquisition speed without averaging. Duringimaging, set the GFP fluorescence signal intensity to below the saturation level by adjusting the detector gain.

5. Adjust the number of prebleach images to five.

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6. Determine the number of images to collect after photobleaching and the time interval between them.

7. Set the laser power to 100% for photobleaching and define the size of a nuclear region to bleach.

8. Check the focal plane and the position of the cell. Begin the FRAP experiment.

Quantitative FRAP Analysis

The following steps involve quantitation and analysis of time-lapse data collected on a Zeiss LSM 510 confocal microscope.LSM 510 image analysis software is used to perform quantitative measurements, and Microsoft Excel is used to calculate andgenerate FRAP recovery curves. During this process, background fluorescence intensity values are subtracted from all measuredvalues, the decrease in the fluorescence intensity due to imaging is normalized, and the corrected prebleach intensity is normal-ized to one. As a result, a FRAP recovery curve-normalized fluorescence intensity as a function of time is generated.

1. Measure the average fluorescence intensity of the bleached region at each time point (IB).

2. Measure the average fluorescence intensity of the entire nucleus at each time point (IN).

3. Measure the average fluorescence intensity of the region outside of cell (background fluorescence intensity) at each timepoint (BB).

4. Measure the average fluorescence intensity of the bleached region during prebleach (IP).

5. Measure the average fluorescence intensity of the entire nucleus during prebleach (INP).

6. Measure the average fluorescence intensity of the region outside of cell (background fluorescence intensity) at each timepoint during prebleach (BP).

7. Calculate the relative fluorescence intensity (IRFI) for each time point using the following formula.

IRFI = (INP − BP) × (IB − BB) / (IN − BB) × (IP − BP)

8. Plot the relative fluorescence intensity for each time point as a function of time.

Note: Data collected at different experiments are combined. Thus, the standard deviation for each time point is calculatedand included in the final FRAP recovery curve.

Notes and Remarks

The current protocol has been extensively used for adherent cell lines. However, it can also be used for cell lines in suspension. Celldensity and metabolic rate are important parameters contributing to the consistency of permeabilization and extraction of cells. It isimperative to process cells that are growing exponentially (in mid-log phase) and are not confluent.

Because enhanced GFP and its variants diffuse relatively freely in the nucleus without substantial binding, they can be used asmarkers for effective permeabilization and extraction. For example, we have found that a GFP chimera containing a single minimalnuclear localization signal sequence (NLS) is highly sensitive to our permeabilization and extraction conditions and is not retainedin the nuclei of the extracted cells. We recommend using cell lines stably expressing GFP-fusion proteins, because the same GFP fu-sion proteins expressed transiently are more susceptible to extraction.

The efficacy of extraction should be assessed by qualitative FRAP analysis in every experiment before proceeding further. This canbe done quickly by observing the mobility of the nuclear protein of interest in live cells (Fig. 1, A and C) and comparing it to themobility observed in permeabilized and extracted cells. If permeabilization and extraction results in the depletion of nuclear mobili-ty factors for the nuclear protein of interest, no fluorescence recovery should be observed (Fig. 1, B and D). Once this is established,purified proteins (for instance, molecular chaperones) or cell extracts can be included in the Recovery Buffer to identify the activityof nuclear mobility factors. Using this method, we have determined that molecular chaperones function as selective steroid receptormobility factors within the nucleus (Fig. 2).

A number of parameters can be examined to assess nuclear integrity after permeabilization and extraction. For example, permeabi-lized cells can be subjected to indirect immunofluorescence analysis to visualize nuclear lamina proteins (such as lamin B or laminC) and heterochromatin-specific proteins (such as HP1α). In this way, the overall integrity of the nuclear envelope and heterochro-

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Fig. 1. FRAP analysis of GFP-GR in nuclei of (A) live or (B) permeabilized and extracted mouse mammary adenocarcinoma cells. Cells weretreated with 100 nM dexamethasone for 1 hour. Single optical z-sections from the mid-planes of cells were acquired before photobleaching andduring recovery at 154 s. A bleached nucleoplasmic region is indicated by a rectangle and is shown as an enlarged pseudocolor image in the lowerpanels. Quantitative FRAP analysis of GFP-GR in (C) live or in (D) permeabilized and extracted cells. Although highly mobile in live cells (A and C),GFP-GR that remains within nuclei of cells after digitonin permeabilization and hypotonic extraction is immobile (B and D). All quantitative data val-ues in FRAP recovery kinetics represent averages ± s.d. from at least 10 cells imaged in three independent experiments. Scale bars, 3 µm.

Fig. 2. Recovery of GR and PR mobility within nuclei of permeabilized and extracted mouse mammary adenocarcinoma cells upon treatmentwith purified molecular chaperones and cochaperones. FRAP analysis of GFP-GR in permeabilized and extracted cells. Cells were treatedwith 100 nM dexamethasone for 1 hour and incubated with combinations of purified molecular chaperone or cochaperone proteins in the pres-ence of ATP. (A) Single optical z-sections from the mid-planes of cells were acquired before photobleaching and during recovery at 154 s. Thebleached nucleoplasmic region is indicated by a rectangle and shown as an enlarged pseudocolor image in the lower panel. (B) QuantitativeFRAP analysis of GFP-GR and GFP-PR-B in permeabilized and extracted 3617.4 and 5953 cells, respectively. 5953 cells were treated with 30nM R5020 for 1 hour. The chaperone mixture in the GFP-GR experiment (A and B) contained Hsp90, Hsp70, p23, p60/Hop, Ydj-1, FKBP51,and CHIP, whereas the chaperone mixture in the GFP-PR-B experiment (B) contained Hsp90, Hsp70, p23, p60/Hop, and Ydj-1. All experi-ments with chaperone mixtures were performed in the presence of ATP. All quantitative data values in FRAP recovery kinetics representmeans ± SD from at least 10 cells imaged in three independent experiments. Scale bar, 3 µm.


matin can be assessed. As a more precise measure of thedifferential extraction of nuclear proteins by the Hypoton-ic Extraction Buffer, we have examined the relative reten-tion of unliganded versus liganded GR after extraction.GFP-GR in hormone treated cells is quite resistant to thecombined digitonin-permeabilization and Hypotonic Ex-traction Buffer extractions. In contrast, unliganded GFP-GR, which is generated by a brief hormone withdrawal, ishighly sensitive to the permeabilization and extractionregimen, in agreement with the low affinity of unligandednuclear GR detected in hormone-withdrawn cells by con-ventional indirect immunofluorescence analysis (16).

Finally, it is important to assess the functional status ofcells after permeabilization and extraction, especially if thecell line of interest has never been used before or substan-tial modifications are introduced to the current protocol.By labeling the sites of active transcription in situ by 5′-bromo-UTP incorporation, one can assess the transcrip-tional status of the permeabilized and extracted cells. Aprotocol for the visualization of transcription sites can befound in (24).

If this method is used to identify the activity of nuclear mobility factors, conditions must be established that dramatically reduce oreven eliminate putative nuclear mobility factors. As shown in Figure 3, the combined digitonin permeabilization and Hypotonic Ex-traction Buffer extraction leads to very efficient loss of chaperone and cochaperone proteins. Thus, if novel nuclear mobility factorsare identified in this assay, the endogenous levels of such mobility factors remaining must be assessed in permeabilized and extract-ed cells.

It is unclear whether the digitonin permeabilization step is absolutely necessary for efficient extraction of nuclear mobility factorsby the Hypotonic Extraction Buffer (16). Importantly, as mentioned in the Introduction, the Hypotonic Extraction Buffer extractionseems necessary to eliminate the requirement for active nuclear transport to restore nuclear mobility factors to the nucleus of perme-abilized cells. In a recently published manuscript (25) from the Nickerson group that described an in vitro FRAP assay, digitoninpermeabilization alone was used to show the ATP-dependent mobility of some RNA splicing factors. It seems likely that proteinmobility factors were not removed from the nucleus in these in vitro assays, highlighting the need for additional extractions, as wehave described, to identify macromolecular components of the nuclear protein mobility machinery.

References and Notes1. G. L. Hager, Studying nuclear receptors with GFP fusions. Methods Enzymol. 302, 73–84 (1999).2. R. Y. Tsien, The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998). 3. R. D. Phair, T. Misteli, High mobility of proteins in the mammalian cell nucleus. Nature 404, 604–609 (2000).4. N. J. McKenna, B. W. O’Malley, Nuclear receptor coactivators—an update. Endocrinology 143, 2461–2465 (2002). 5. A. Aranda, A. Pascual, Nuclear hormone receptors and gene expression. Physiol. Rev. 81, 1269–1304 (2001).6. C. K. Glass, M. G. Rosenfeld, The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 14, 121–141 (2000).7. L. P. Freedman, Increasing the complexity of coactivation in nuclear receptor signaling. Cell 97, 5–8 (1999).8. V. V. Ogryzko, R. L. Schiltz, V. Russanova, B. H. Howard, Y. Nakatani, The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87,

953–959 (1996). 9. T. E. Spencer, G. Jenster, M. M. Burcin, C. D. Allis, J. Zhou, C. A. Mizzen, N. J. McKenna, S. A. Onate, S. Y. Tsai, M. J. Tsai, B. W. O’Malley, Steroid receptor

coactivator-1 is a histone acetyltransferase. Nature 389, 194–198 (1997).10. H. Chen, R. J. Lin, W. Xie, D. Wilpitz, R. M. Evans, Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acety-

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Fig. 3. Chaperone and cochaperone proteins remaining in permeabi-lized and extracted mouse mammary adenocarcinoma cells stably ex-pressing GFP-GR. Cells were treated with 100 nM dexamethasone for1 hour. Total protein was extracted from intact (Total) or permeabilizedand extracted cells (Perm). Expression of endogenous Hsp90, Hsp70,p60/Hop, Hsp40, p23, lamin B, and GR was detected by Western blotanalysis using specific antibodies.


20. H. Kosano, B. Stensgard, M. C. Charlesworth, N. McMahon, D. Toft, The assembly of progesterone receptor-hsp90 complexes using purified proteins. J. Biol.Chem. 273, 32973–32979 (1998).

21. K. D. Dittmar, W. B. Pratt, Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70-dependent step is sufficient for creating the steroid binding conformation. J. Biol. Chem. 272, 13047–13054 (1997).

22. C. Elbi, T. Misteli, G. L. Hager, Recruitment of dioxin receptor to active transcription sites. Mol. Cell. Biol. 13, 2001–2015 (2002).23. M. Dundr, T. Misteli, in Current Protocols in Cell Biology, Chapter 13: Organelle Motility, Unit 13.5, Measuring Dynamics of Nuclear Proteins by Photobleaching.

John Davey, Mike Lord, Eds. (Wiley and Sons, New York, 2004).24. J. Ellenberg, J. Lippincott-Schwartz, in Cells, A Laboratory Manual, Volume 2: Microscopy and Cell Structure, Chapter 79. Fluorescence Photobleaching Tech-

niques. David L. Spector, Robert D. Goldman, Leslie A. Leinwand, Eds. (Cold Spring Harbor Laboratory Press, Plainview, NY, 1998). 25. S. Wagner, S. Chiosea, M. Ivshina, J. A. Nickerson, In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein

SRm160. J. Cell Biol. 164, 843–850 (2004). 26. We thank P. Badger for technical assistance on FRAP analysis and B. Stesgard for help with protein purification. Imaging was carried out in the Fluorescence

Imaging Facility, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute.

Citation: C. Elbi, D. A. Walker, M. Lewis, G. Romero, W. P. Sullivan, D. O. Toft, G. L. Hager, D. B. DeFranco, A novel in situ assay for the identificationand characterization of soluble nuclear mobility factors. Sci. STKE 2004, pl10 (2004).

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